This present invention relates to calcium channel compositions. In particular, this invention relates to a mammalian gene encoding a retinal calcium channel subunit polypeptide, herein referred to as CACNA1F, wherein mutations of CACNA1F may cause a type of X-linked congenital stationary night blindness.
The following references are cited in the application as numbers in brackets ([ ]) at the relevant portion of the application. Each of these references is incorporated herein by reference.
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X-linked congenital stationary night blindness (CSNB) is a non-progressive retinal disorder characterized by night blindness, decreased visual acuity, myopia, nystagmus and strabismus [1]. Two distinct clinical entities of CSNB have been proposed, complete and incomplete CSNB [2]. In patients with complete CSNB, rod function is not detectable, whereas patients with incomplete CSNB have reduced, but not extinguished rod function. Furthermore, patients with complete CSNB may show moderate to severe myopia, whereas those with incomplete CSNB may show severe myopia to hyperopia [1]. A related disorder, Aaland Island eye disease (AIED), is suggested to be clinically indistinguishable from incomplete CSNB [3].
The biochemical defects underlying complete and incomplete CSNB are not known, but may be revealed by identifying the genes involved in these disorders. The CSNB gene(s) has been localized to the short arm of the human X-chromosome to region p11 by linkage analysis. However, it was uncertain whether the phenotypic variation results from genetic heterogeneity or a single locus exhibiting a wide variation in clinical phenotype [4]. Studies of three families with AIED have localized the AIED gene between DXS7 DXS255, overlapping with the chromosomal region harbouring the gene for incomplete CSNB.
Calcium channels are membrane-spanning hetero-oligomeric protein complexes, consisting of (alpha)1, (alpha)2, (beta)1, (beta)2, delta and gamma subunits [5], that allow controlled entry of Ca2+ ions into the cytoplasm from the extracellular space or from intracellular stores. All cells throughout the animal kingdom and some plant, bacteria and fungal cells possess one or more types of calcium channel, which play a central role in the regulation of intracellular Ca2+ concentration. Changes in intracellular Ca2+ concentration are implicated in a number of vital processes, such as neurotransmitter release, muscle contraction, pacemaker activity and the secretion of hormones and other substances.
Voltage-gated calcium channels (types L, N, and P) are located on the plasma membrane of all excitable animal cells, such as neurons and muscle cells. L-type voltage-gated channels are distinguished pharmacologically from the other types by, among other features, their ability to bind dihydropyridine.
The (alpha)1-subunits of L-type channels function as the pore and voltage sensors in calcium ion-selective pores [5]. Several diseases are known to be the result of mutations in calcium channel (alpha)1-subunit genes, including human familial hemiplegic migraine and episodic ataxia type-2, hypokalemic periodic paralysis, muscular dysgenesis (mdg) and absence epilepsy in tottering mice. Mutations in an L-type calcium channel (alpha)1-subunit gene cause myotonia in C. elegans, and a non L-type calcium channel (alpha)1-subunit gene in Drosophilia (DmcalA) is a suggested candidate gene for the night-blind-A (nbA) and cacophony (cac) mutations.
Patients with CSNB, both complete and incomplete, show a reduced b-wave response on electroretinographic testing and decreased dark adaptation. Light-induced hyperpolarization of photoreceptor cells diminishes the release of neurotransmitters at their synaptic terminals, which in turn leads to the depolarization of outer nuclear bipolar and horizontal cells. This depolarization of bipolar cells causes the subsequent depolarization of Mueller cells, which appears largely to be the origin of the corneal positive b-wave [6]. The influx of calcium through dihydropyridine-sensitive calcium channels into photoreceptor cells has been shown to mediate the release of neurotransmitter [7]. Therefore, it is reasonable to presume that one or more L-type voltage-gated channels is involved in neurotransmission in the eye.
High-density physical maps of the Xp11.23 cytogenetic region have been constructed in YACs [8], cosmids [9], and BACs [10]. Large scale DNA sequencing in the Xp11.23 region has revealed several new genes. Computer analysis (GRAIL(trademark) and GENE-ID(trademark)) of an extended genomic DNA sequence within the Xp11.23 region, has identified potential exons with homology to calcium channel (alpha)1-subunit genes [28]. There was an indication that this gene was expressed in skeletal muscle, but this assertion may not be supported by the reported data [28]. The HUGO/GDB Nomenclature Committee has assigned this putative gene the name CACNA1F. The same putative gene was identified by the GENSCAN(trademark), in a computer search of about 1,000 Kb of genomic DNA in this region (Xp11.23) by the Genome Sequencing Centre, Jena.
The identification of the gene which is causative of incomplete CSNB may allow for development of diagnostic tests for this disorder and risk assessment in affected families. As well, identification of the gene which is causative of incomplete CSNB will provide information as to the basic defect in this retinal condition, which could lead to effective methods for treatment or cure of the disorder. Furthermore, as the associated features of myopia, nystagmus and strabismus frequently observed in patients with incomplete CSNB may possibly be caused by calcium-regulated developmental pathways, identification of the retinal calcium channel gene may help to elucidate the molecular details of eye development and which may lead to treatment for related eye disorders or diseases.
The identification of a calcium channel gene that is expressed in the human retina. will aid in the elucidation of the role of calcium channels in retinal function. Knowledge of the structure of this gene will lead to studies of the structure-function relationships of the protein in the retinal environment. This knowledge, in turn, would be useful in the design and discovery of therapeutic agents whose activities are exerted by interacting directly or indirectly with a calcium channel.
Finally, given the diversity and importance of voltage-gated calcium channels in mammalian physiology, possession of cells which express selected channel subtypes would find use in the area of pharmacology and drug design. The identification of novel channel subtypes will expand this area of the medical arts.
The region on the short arm of the human X-chromosome, Xp11, which carries the gene for incomplete CSNB (CSNB2) was refined to a distance of 1.2 Mb, between DXS722 and DXS255.
A gene, CACNA1F, with homology to voltage-gated L-type calcium channel (alpha)1-subunit genes, and that mapped to the CSNB2 minimal region was identified as being retina-specific. The complete cDNA sequence of this gene has been elucidated. Mutational analysis of CACNA1F in 31 families with incomplete CSNB revealed 15 different mutations, predicted to cause premature termination of, or missense mutations in, the protein product of CACNA1F. Together, these findings establish that mutations in CACNA1F cause incomplete CSNB.
In addition, mutational analysis of CACNA1F in four families with AIED revealed four different mutations, predicted to cause premature termination of, or missense mutations in, the protein product of CACNA1F. Two mutations were found in both a family diagnosed with incomplete CSNB and another family diagnosed with AIED, suggesting that these two disorders are the same. Therefore, in total, mutational analysis of 35 families with either incomplete CSNB or AIED revealed 17 mutations in the CACNA1F gene.
The murine orthologue of the human CACNA1F has been identified and the cDNA sequence determined. There is a high degree of sequence homology between the murine and the human CACNA1F gene, which is as high as 95% in some regions.
The present invention provides a mammalian nucleic acid sequence encoding a novel calcium channel (alpha)1F-subunit expressed in the retina. Thus, in one aspect, this invention is an isolated DNA molecule comprising a sequence of nucleotides that encodes an (alpha)1F-subunit of a mammalian retinal calcium channel, including a human (alpha)1F-subunit, a murine (alpha)1F-subunit and orthologues of the human and murine (alpha)1F-subunits.
In one embodiment, the invention comprises a DNA molecule that encodes a human retinal (alpha)1F-subunit and has a sequence of nucleotides selected from a group consisting of:
(a) the sequence set forth in SEQ ID NO. 1;
(b) a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO 2;
(c) the sequence set forth in SEQ ID NO. 3; or
(d) a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO 4.
In another embodiment, this invention comprises a DNA molecule that encodes a murine (alpha)1F-subunit and has a sequence of nucleotides selected from a group consisting of:
(a) the sequence set forth in SEQ ID NO 5; or
(b) a sequence of nucleotides that encodes the sequence of amino acids set forth in SEQ ID NO6.
In another aspect, this invention comprises a substantially pure (alpha)1F-subunit of a mammalian retinal calcium channel, including a human (alpha)1F-subunit represented by the sequence of amino acids set forth in SEQ ID NO. 2 or 4, a murine (alpha)1F-subunit represented by the sequence of amino acids set forth in SEQ ID NO. 6, and orthologues of the human and murine (alpha)1F-subunits.
In another aspect, this invention comprises an isolated RNA sequence that encodes an (alpha)1F-subunit of a mammalian retinal calcium channel or an antisense RNA molecule having a sequence that is complementary to the mRNA encoding an (alpha)1F-subunit of a mammalian retinal calcium channel.
In another aspect, this invention comprises an expression vector, preferably a mammalian expression vector, comprising the nucleotide sequence of an (alpha)1F-subunit of a mammalian retinal calcium channel.
In another aspect, this invention is a cell, preferably a eukaryotic cell, comprising a heterologous DNA comprising a nucleotide sequence of (alpha)1F-subunit of a mammalian retinal calcium channel.
In another aspect, this invention comprises an isolated nucleic acid that encodes a full-length (alpha)1F-subunit of a mammalian retinal calcium channel, wherein the nucleic acid molecule is fully complementary to nucleic acid which is native to a mammalian retinal cell.
In another aspect, the invention comprises a method of diagnosing incomplete CSNB which method includes screening for alterations in the sequence of nucleotides disclosed herein.